The mechanism of superoxide (O2-) production in the macula densa is not known. We will test our hypotheses with the following specific aims. Aim 1: Hypothesis: The increase in tubular NaCI concentration that initiates tubuloglomerular feedback enhances O2- production primarily from macula densa NAD(P)H oxidase (NOX). We will measure O2- production while increasing luminal NaCI in macula densa. To investigate the Nox isoform expressed at the macula densa, we will isolate macula densa cells using laser capture Microdissection (LCM) and real time PCR. Aim 2: Hypothesis: O2- production is activated by macula densa depolarization. Depolarization of the macula densa activates NAD(P)H oxidase by stimulating the GTP-binding protein Rac. We will measure O2- generation while: a) depolarizing the macula densa;and b) using dominant negative Rac and constitutively active Rac expressed macula densa cells. Aim 3: Hypothesis: O2- production is enhanced by increased macula densa intracellular pH, which process is involved in transporting protons generated during O2- production out of the macula densa by Na/H exchange and/or H/K ATPase. We will measure O2- production while increasing luminal NaCI: a) in the presence and absence of H/K ATPase inhibitors;and b) while clamping intracellular pH. We will measure O2- generation while increasing luminal NaCI in H/K ATPase knockout mice. Aim 4 Hypothesis: Activity of apical Na/H exchangers is higher in SHR than in WKY, resulting in higher macula densa pH, which enhances NAD(P)H oxidase activity and superoxide production and thus augments tubuloglomerular feedback. Inhibition of macula densa apical Na/H exchange tends to normalize tubuloglomerular feedback in SHR. We will study the activity of apical and basolateral Na/H exchangers at the macula densa in SHR and WKY. We will isolate the apical and basolateral membranes of the macula densa using LCM and study the Na/H exchanger expression level using real-time PCR. We will measure tubuloglomerular feedback in SHR while clamping macula densa intracellular pH to the same degree as WKY. In present research, we will study the mechanism and effect of superoxide on renal hemodynamic and blood pressure. We believe the results of the present study will help us better understand the causes of hypertension and might reveal new approaches for treatment.